Scytovirin domain 1 related polypeptides

ABSTRACT

A scytovirin domain 1 (SD1) polypeptide, a nucleic acid encoding the polypeptide, and related fusion proteins, conjugates, isolated cells, vectors, and antibodies, as well as a method of inhibiting a viral infection using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicantSer. No. 11/914,833, filed Dec. 7, 2007, now U.S. Pat. No. 8,067,530,which is the U.S. National Phase of International Patent Application No.PCT/U.S. 06/20100, filed May 24, 2006, now WO 2006/127822, which claimsthe benefit of U.S. Provisional Patent Application No. 60/684,353, filedMay 25, 2005, all of which are hereby incorporated by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 2,998 Byte ASCII (Text) file named“708802SeqListing ST25.txt” created on Oct. 17, 2011.

BACKGROUND OF THE INVENTION

Viral infections remain among the most formidable causes of human andnon-human animal morbidity and mortality worldwide. Effectivepreventions or therapies against most viral pathogens remain elusive.One of the most contemporary and catastrophic examples is the stillrapidly expanding and pervasive worldwide pandemic of HIV (humanimmunodeficiency virus) infection and AIDS (acquired immune deficiencysyndrome). Despite more than two decades of research to find effectivepreventative or therapeutic vaccines or drugs, surprisingly littleprogress has been made. The need for new effective preventative andtherapeutic agents for HIV/AIDS and other potentially lethal viraldiseases remains an urgent global priority.

Peptidic molecules offer tremendous structural diversity that can beexploited for development of novel therapeutics and preventions of manydifferent kinds of diseases. For example, in the field of HIVtherapeutics a novel, rationally-constructed peptide molecule known asT-20 (Kilby, Nat. Med., 4: 1302-1307 (1998)) has been recently shown tobe a potent inhibitor of HIV/cell fusion. Furthermore, naturallyoccurring, non-mammalian peptides and proteins offer new avenues forantiviral discovery and development. An outstanding example is theremarkable HIV-inactivating protein cyanovirin-N (Boyd et al.,Antimicrob. Agents Chemother., 41: 1521-1530 (1997)). Additionally,International Published Application WO 03/097814 (Boyd et al.) andBokesch et al. (Biochem., 42: 2578-2584 (2003)) disclose the discoveryof antiviral scytovirin. Clearly, there is great untapped potential fordiscovery and development of novel, polypeptides and proteins that canbe used in prevention and therapeutics of viral diseases.

The present invention provides new antiviral polypeptides and proteins,fusion proteins, and conjugates, as well as nucleic acids, vectors, hostcells, and related compositions and methods of use thereof to inhibitviral infections. These and other aspects and advantages of the presentinvention, as well as additional inventive features, will becomeapparent from the description provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a polypeptide comprising an amino acid sequencethat has 65% or greater sequence identity to SEQ ID NO: 1, provided thatthe polypeptide does not comprise SEQ ID NO: 4. The invention alsoprovides a fusion protein and conjugate comprising the polypeptide, aswell as a nucleic acid comprising a nucleic acid sequence encoding thepolypeptide and an isolated cell comprising the nucleic acid.

The invention further provides an antibody that binds to thepolypeptide, wherein the antibody binding site is part of the amino acidsequence that has 65% or greater sequence identity to SEQ ID NO: 1. Acomposition comprising (i) the polypeptide, fusion protein, conjugate,nucleic acid, isolated cell, or antibody, and (ii) a carrier, excipient,or adjuvant therefore, also is provided herein.

The invention provides methods of using the above compounds andcompositions for inhibiting a viral infection. One aspect of theinvention provides a method of inhibiting a viral infection in a host,which method comprises administering to the host a viralinfection-inhibiting amount of at least one member of the groupconsisting of the (i) polypeptide, (ii) fusion protein, (iii) conjugate,(iv) nucleic acid, (v) isolated cell, (vi) antibody, or (vii)composition of the invention, which method, optionally, furthercomprises the prior, simultaneous, or subsequent administration, by thesame route or a different route, of a substance other than (i)-(vii)that is efficacious in inhibiting the viral infection, whereupon theviral infection is inhibited.

Another aspect of the invention provides a method of inhibiting theinfection of a host by a virus, which method comprises administering tothe host the antibody of the invention in an amount sufficient to inducein the host an immune response to the virus, which method, optionally,further comprises the prior, simultaneous, or subsequent administration,by the same or a different route, of a substance other than the antibodythat is efficacious in inhibiting the virus or inducing an immuneresponse to the virus, whereupon the infection of the host by the virusis inhibited.

In yet another aspect, the invention provides a method of inhibiting avirus in a biological sample or in/on an inanimate object, which methodcomprises contacting the biological sample or the inanimate object witha viral-inhibiting amount of at least one of the (i) polypeptide, (ii)fusion protein, or (iii) conjugate of the invention. The method,optionally, further comprises the prior, simultaneous, or subsequentcontacting, in the same manner or in a different manner, of thebiological sample or inanimate object with a substance other than(i)-(iii) that is efficacious in inhibiting the virus, whereupon thevirus is inhibited.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 is a graph of absorbance at 450 nm versus concentration (μg/mL)of scytovirin (▴) or SD1 [scy(1-48)] (+), which illustrates scytovirinand SD1 binding to HIV-1 gp41 ectodomain.

FIG. 2 is a graph of absorbance at 450 nm versus concentration (μg/mL)of scytovirin (▴) or SD1 [scy(1-48)] (+), which illustrates scytovirinand SD1 binding to HIV-1 gp120.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptide, Fusion Protein, and Conjugate

The invention provides a polypeptide comprising an amino acid sequencethat has 65% or greater sequence identity to SEQ ID NO: 1, provided thatthe polypeptide does not comprise SEQ ID NO: 4. SEQ ID NO: 4 sets forththe amino acid sequence of the scytovirin polypeptide and SEQ ID NO: 1sets forth an amino acid sequence derived from the scytovirinpolypeptide that has biological properties similar to the scytovirinpolypeptide. In particular, SEQ ID NO: 1 is derived from amino acids1-48 of scytovirin, which is referred to herein as scytovirin domain 1(SD1). Accordingly, SEQ ID NO: 1 (e.g., a polypeptide consisting of SEQID NO: 1) is therefore referred to as SD1. The polypeptide of theinvention is based on a portion of the scytovirin polypeptide (SEQ IDNO: 4) that has antiviral properties. In this regard, the polypeptide ofthe invention desirably has the ability to bind viral proteins (e.g.,gp41 or gp120 of an immunodeficiency virus (e.g., HIV)) and/or exhibitantiviral activity against HIV or other viruses (Ebola, influenza,etc.). More preferably, the polypeptide of the invention has biologicalaffinity for viral proteins such as gp41 or gp120, or antiviralactivity) comparable to or greater than that of scytovirin.

“Antiviral” or “antiviral activity” as used herein, both with respect tothe polypeptide of the invention and other components or compositions ofthe invention described herein, refers to the ability of a compound toinhibit a virus. Inhibition of a virus encompasses slowing or stoppingthe rate of replication or infection of a virus (e.g., within anorganism), reducing the infectivity of the virus (e.g., HIV), orinhibiting or preventing the virus from invading a host (e.g., byinducing an immune response in the host). Thus antiviral activityencompasses the activity of a substance or molecule directly against thevirus (e.g., by binding viral proteins) or indirectly (e.g., by inducingan immune response). Antiviral activity can be demonstrated by anysuitable method, such as by in vitro antiviral assays (e.g., Gulakowskiet al., J. Virol. Methods, 33: 87-100 (1991), which accurately predictfor antiviral activity in humans. Such assays measure the ability ofcompounds to prevent the replication of HIV and/or the cytopathiceffects of HIV on human target cells. These measurements directlycorrelate with the pathogenesis of HIV-induced disease in vivo.

The terms “sequence identity” and “percent identity” of a sequence(amino acid or nucleic acid sequence), as used herein, means the percentof amino acids that are identical between two optimally alignedsequences. Optimal alignment can be calculated by any of several knownalgorithms. For the purposes of the invention, the sequence identity isdetermined using the well-known Basic Local Alignment Search Tool(BLAST), which is publicly available through the National CancerInstitute/National Institutes of Health (Bethesda, Md.) and has beendescribed in printed publications (see, e.g., Altschul et al., J. Mol.Biol., 215(3), 403-10 (1990)).

The polypeptide of the invention can comprise an amino acid sequencethat has 65% or greater sequence identity to SEQ ID NO: 1, provided thatthe polypeptide does not comprise the amino acid sequence of SEQ ID NO:4. However, it is preferred that the polypeptide comprises an amino acidsequence that has a higher percent identity to SEQ ID NO: 1, forexample, an amino acid sequence that has 70% or greater (e.g., 75% orgreater), more preferably 80% or greater (e.g., 85% or greater) or even90% or greater (e.g., 95%, 96%, 97%, 98%, or 99%) sequence identity toSEQ ID NO: 1, provided that the polypeptide does not have the amino acidsequence of SEQ ID NO: 4. It is especially preferred that thepolypeptide comprises the amino acid sequence of SEQ ID NO: 2, and evenmore preferred that the polypeptide comprises the amino acid sequence ofSEQ ID NO: 1. The polypeptide can comprise D-amino acids, L-amino acidsor a mixture of D- and L-amino acids. The D-form of the amino acids,however, is particularly preferred, since a protein comprised of D-aminoacids is expected to have a greater retention of its biological activityin vivo, given that the D-amino acids are not recognized by naturallyoccurring proteases.

Accordingly, the polypeptide of the invention can be a fragment orvariant of the polypeptide of SEQ ID NO: 1 (e.g., a polypeptidecomprising an amino acid sequence having the aforementionedpercent-identity to SEQ ID NO: 1). Such fragments or variants cancomprise deletions or substitutions of the amino acid sequence, providedthat such deletions and substitutions do not destroy the usefulness ofthe polypeptide as described herein. The variants encompassed by theinvention can comprise, for example, (i) one or more conservative orneutral amino acid substitutions (e.g., 1-20, preferably 1-10, morepreferably 1, 2, 3, 4, or 5) and/or (ii) 1-20, preferably 1-10, morepreferably 1, 2, 3, 4 or 5, and even more preferably, 1, 2, or 3, aminoacid additions at the N-terminus and/or the C-terminus, with the provisothat the variant has activity characteristic of a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 1 (e.g., antiviral activity) to agreater or lesser extent but not negated. Preferably, the amino acid atposition 7 of SEQ ID NO: 1 is not substituted with cysteine. Similarly,fragments of SEQ ID NO: 1 can be any fragment that retains the functionof a polypeptide comprising SEQ ID NO: 1 to a greater or lesser extent,but not negated. Such fragments preferably comprise about 5 or morecontiguous amino acids (e.g., about 5-30 contiguous amino acids) orabout 10 or more contiguous amino acids (e.g., about 15-25 contiguousamino acids) of SEQ ID NO: 1.

Alterations of the amino acid sequence of SEQ ID NO: 1 to producefragment or variant polypeptides can be done by a variety of methodsknown to those skilled in the art. For instance, amino acidsubstitutions can be conveniently introduced into the proteins at thetime of synthesis. Alternatively, site-specific mutations can beintroduced by ligating into an expression vector a synthesizedoligonucleotide comprising the modified site. Alternatively,oligonucleotide-directed, site-specific mutagenesis procedures can beused, such as disclosed in Walder et al., Gene, 42: 133 (1986); Bauer etal., Gene 37: 73 (1985); Craik, Biotechniques, 12-19 (1995); and U.S.Pat. Nos. 4,518,584 and 4,737,462.

It is within the skill of the ordinary artisan to select synthetic andnaturally-occurring amino acids that effect conservative or neutralsubstitutions for any particular amino acids in a given amino acidsequence (e.g., SEQ ID NO: 1). The ordinarily skilled artisan desirablywill consider the context in which any particular amino acidsubstitution is made, in addition to considering the hydrophobicity orpolarity of the side-chain, the general size of the side chain and thepK value of side-chains with acidic or basic character underphysiological conditions. For example, lysine, arginine, and histidineare often suitably substituted for each other, and more often arginineand histidine are suitably substituted for each other. As is known inthe art, this is because all three amino acids have basic side chains,whereas the pK value for the side-chains of lysine and arginine are muchcloser to each other (about 10 and 12) than to histidine (about 6).Similarly, glycine, alanine, valine, leucine, and isoleucine are oftensuitably substituted for each other, with the proviso that glycine isfrequently not suitably substituted for the other members of the group.This is because each of these amino acids are relatively hydrophobicwhen incorporated into a polypeptide, but glycine's lack of an α-carbonallows the phi and psi angles of rotation (around the α-carbon) so muchconformational freedom that glycinyl residues can trigger changes inconformation or secondary structure that do not often occur when theother amino acids are substituted for each other. Other groups of aminoacids frequently suitably substituted for each other include, but arenot limited to, the group consisting of glutamic and aspartic acids; thegroup consisting of phenylalanine, tyrosine and tryptophan; and thegroup consisting of serine, threonine, and, optionally, tyrosine.Additionally, the ordinarily skilled artisan can readily group syntheticamino acids with naturally-occurring amino acids.

The polypeptide of the invention can be prepared by any of a number ofconventional techniques. The polypeptide can be isolated or purifiedfrom a naturally occurring source or from a recombinant source. Suitabletechniques for producing the recombinant polypeptides are known (forgeneral background see, e.g., Nicholl, in An Introduction to GeneticEngineering, Cambridge University Press: Cambridge (1994), pp. 1-5 &127-130; Steinberg et al., in Recombinant DNA Technology Concepts andBiomedical Applications, Prentice Hall: Englewood Cliffs, N.J. (1993),pp. 81-124 & 150-162; Sofer, in Introduction to Genetic Engineering,Butterworth-Heinemann, Stoneham, Mass. (1991), pp. 1-21 & 103-126; Oldet al., in Principles of Gene Manipulation, Blackwell ScientificPublishers: London (1992), pp. 1-13 & 108-221; and Emtage, in DeliverySystems for Peptide Drugs, Davis et al., eds., Plenum Press: New York(1986), pp. 23-33). For instance, a nucleic acid encoding a desiredprotein can be subcloned into an appropriate vector using well-knownmolecular genetic techniques (see, e.g., Maniatis et al., MolecularCloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory,1989)). The nucleic acid can be transcribed and the protein subsequentlytranslated in vitro. Commercially available kits also can be employed(e.g., such as manufactured by Clontech, Palo Alto, Calif.; AmershamLife Sciences, Inc., Arlington Heights, Ill.; InVitrogen, San Diego,Calif., and the like).

Such polypeptides also can be synthesized using an automated peptidesynthesizer in accordance with methods known in the art. Alternatively,the polypeptide can be synthesized using standard peptide synthesizingtechniques well-known to those of skill in the art (e.g., as summarizedin Bodanszky, Principles of Peptide Synthesis, (Springer-Verlag,Heidelberg: 1984)). In particular, the polypeptide can be synthesizedusing the procedure of solid-phase synthesis (see, e.g., Merrifield, J.Am. Chem. Soc., 85: 2149-54 (1963); Barany et al., Int. J. PeptideProtein Res., 30: 705-739 (1987); and U.S. Pat. No. 5,424,398). Ifdesired, this can be done using an automated peptide synthesizer.Removal of the t-butyloxycarbonyl (t-BOC) or9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups andseparation of the polypeptide from the resin can be accomplished by, forexample, acid treatment at reduced temperature. Thepolypeptide-containing mixture then can be extracted, for instance, withdiethyl ether, to remove non-peptidic organic compounds, and thesynthesized polypeptide can be extracted from the resin powder (e.g.,with about 25% w/v acetic acid). Following the synthesis of thepolypeptide, further purification (e.g., using HPLC) optionally can bedone in order to eliminate any incomplete proteins, polypeptides,peptides or free amino acids. Amino acid and/or HPLC analysis can beperformed on the synthesized polypeptide to validate its identity.

The overall size of the polypeptide of the invention is not particularlylimited. The polypeptide can consist of or consist essentially of, anyone or more of the sequences or fragments described herein, in whichcase the overall size of the polypeptide will correspond, substantiallyor exactly to the one or more particular sequences or fragments of theinvention that are used. Alternatively, the polypeptide will compriseany one or more of the sequences or fragments described herein alongwith appropriate flanking sequences. Appropriate flanking sequences arethose that do not interfere with the function of the polypeptide, andcan be selected in accordance with general principals of peptide designknown in the art. In this respect, the polypeptide typically willcomprise about 1000 or fewer amino acids, such as about 500 or fewer,about 400 or fewer, about 300 or fewer, about 200 or fewer, about 150 orfewer, about 100 or fewer, or even about 50 or fewer amino acids.

Two or more polypeptides of the invention can be linked by a flexiblelinker. The flexible linker can be any suitable peptide or othermolecule or composition that can be used to join two or more polypeptidedomains. Peptide linkers preferably comprises about 5 or more aminoacids (e.g., about 6 or more, 7 or more, or 9 or more amino acids), morepreferably about 10 or more amino acids (e.g., about 11 or more, 12 ormore, or 14 or more amino acids), and most preferably about 15 or moreamino acids (e.g., about 17 or more, 20 or more, or 25 or more aminoacids). Linker sequences, as well as methods for joining polypeptidedomains using flexible linkers, are known in the art (see, e.g.,Imanishi et al., Biochem. Biophys. Res. Commun., 333(1): 167-173 (2005);Lin et al., Eur. Cytokine Netw., 15(3): 240-246 (2004)). An exemplarylinker sequence is GGGGSGGGGSGGGGS (SEQ ID NO: 5).

The polypeptide of the invention can be joined to other biomolecules,such as, for example, proteins, polypeptides, lipids, carbohydrates,prenyl, and acyl moieties, and nucleic acids. For example, it may bepreferable to produce the protein as part of a larger fusion protein,either by chemical conjugation, or through genetic means, such as areknown to those skilled in the art. In this regard, the invention alsoprovides a fusion protein comprising the polypeptide of the inventionand one or more other protein(s) having any desired properties oreffector functions, such as cytotoxic or immunological properties, orother desired properties, such as to facilitate isolation, purification,or analysis of the fusion protein. For example, effector components foruse in the fusion polypeptide include immunological reagents and toxins.Immunological reagent refers to an antibody, an immunoglobulin, or animmunological recognition element. An immunological recognition elementis an element, such as a peptide (e.g., a FLAG octapeptide leadersequence) that can be appended to make a recombinant SD1-FLAG fusionprotein, wherein the FLAG element facilitates, through immunologicalrecognition, isolation and/or purification and/or analysis of thepolypeptide (or fragment thereof) or variant thereof to which it isattached. A toxin can be any suitable toxin, such as Pseudomonasexotoxin. The addition of a toxin or immunological reagent to thepolypeptide of the invention can facilitate purification and analysis ofthe polypeptide (e.g., such as a FLAG-SD1 fusion protein) or forspecific targeting to a virus or viral-infected cells (e.g., HIV and/orHIV-infected cells). In these instances, the polypeptide of theinvention serves not only as antiviral or as a neutralizing agent butalso as a targeting agent to direct the effector activities of the otherproteins selectively against a given virus, such as HIV. Thus, forexample, a therapeutic agent can be obtained by combining theHIV-targeting function of the polypeptide of the invention with a toxinaimed at neutralizing an infectious virus and/or by destroying cellsproducing an infectious virus, such as HIV. Similarly, a therapeuticagent can be obtained, which combines the viral-targeting function of apolypeptide of the invention with the multivalency and effectorfunctions of various immunoglobulin subclasses. A preferred fusionprotein comprises the polypeptide of the invention and albumin.

The generation of fusion proteins is within the ordinary skill in theart (see, e.g., Chaudhary et al. (1988), supra) and can involve the useof restriction enzyme or recombinational cloning techniques (see, e.g.,Gateway™ (Invitrogen, Carlsbad, Calif.)). See, also, U.S. Pat. No.5,314,995. In a transcriptional gene fusion, the DNA or cDNA willcontain its own control sequence directing appropriate production ofprotein (e.g., ribosome binding site, translation initiation codon,etc.), and the transcriptional control sequences (e.g., promoterelements and/or enhancers) will be provided by the vector. In atranslational gene fusion, transcriptional control sequences as well asat least some of the translational control sequences (i.e., thetranslational initiation codon) will be provided by the vector. In thecase of a translational gene fusion, a chimeric protein will beproduced.

If desired, the polypeptide or fusion protein of the invention can bemodified, for instance, by glycosylation, amidation, carboxylation, orphosphorylation, or by the creation of acid addition salts, amides,esters, in particular C-terminal esters, and N-acyl derivatives of theproteins of the invention. The polypeptide or fusion protein also can bemodified to create protein derivatives by forming covalent ornoncovalent complexes with other moieties in accordance with methodsknown in the art. Covalently-bound complexes can be prepared by linkingthe chemical moieties to functional groups on the side chains of aminoacids comprising the proteins, or at the N- or C-terminus. Desirably,such modifications and conjugations do not adversely affect the activityof the polypeptide or fusion protein. While such modifications andconjugations can increase or reduce activity, the activity desirably isnot negated and is characteristic of the unaltered polypeptide.

The invention provides a conjugate comprising a polypeptide of theinvention and one or more effector components. Conjugates (e.g.,viral-targeted conjugates) can be prepared by chemical coupling of thepolypeptide of the invention targeting component with the effectorcomponent. The most feasible or appropriate technique to be used toconstruct a given conjugate will be selected based upon consideration ofthe characteristics of the particular effector molecule selected. Forexample, with a selected non-proteinaceous effector component, chemicalcoupling may be the only feasible option for creating the desiredconjugate.

Examples of effector components or other functional reagents suitablefor chemical coupling to the polypeptide of the invention and therebyused as effector components in the inventive conjugates can include, forexample, polyethylene glycol, dextran, an antiviral agent (other than apolypeptide), a solid support matrix, and the like, whose intendedeffector functions can include one or more of the following: to improvestability of the conjugate; to increase the half-life of the conjugate;to increase resistance of the conjugate to proteolysis; to decrease theimmunogenicity of the conjugate; to attach or immobilize a polypeptideof the invention onto a solid support matrix (i.e., in such instance thesolid support matrix can be the effector component of the conjugate)(e.g., see, for example, Harris, in Poly(Ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, Harris, ed., Plenum Press: NewYork (1992), pp. 1-14). The antiviral agent can be any suitableantiviral agent, including AZT, ddA, ddI, ddC, 3TC gancyclovir,fluorinated dideoxynucleosides, acyclovir, α-interferon, nonnucleosideanalog compounds, such as nevirapine (Shih et al., PNAS, 88: 9878-9882,(1991)), TIBO derivatives, such as R82913 (White et al., Antiviral Res.,16: 257-266 (1991)), Ro31-8959, BI-RJ-70 (Merigan, Am. J. Med., 90(Supp1.4A): 8S-17S (1991)), michellamines (Boyd et al., J. Med. Chem.,37: 1740-1745 (1994)) and calanolides (Kashman et al., J. Med. Chem.,35: 2735-2743 (1992)), nonoxynol-9, gossypol and derivatives,gramicidin, cyanovirin-N and functional homologs thereof (Boyd et al.(1997), supra). Other exemplary antiviral compounds include proteaseinhibitors (see R. C. Ogden and C. W. Flexner, eds., Protease Inhibitorsin AIDS Therapy, Marcel Dekker, N.Y., 2001), such as saquinavir (see I.B. Duncan and S. Redshaw, in R. C. Ogden and C. W. Flexner, supra, pp.27-48), ritonavir (see D. J. Kempf, in R. C. Ogden and C. W. Flexner,supra, pp. 49-64), indinavir (see B. D. Dorsey and J. P. Vacca, in R. C.Ogden and C. W. Flexner, supra, pp. 65-84), nelfinavir (see S. H. Reich,in R. C. Ogden and C. W. Flexner, supra, pp. 85-100), amprenavir (see R.D. Tung, in R. C. Ogden and C. W. Flexner, supra, pp. 101-118), andanti-TAT agents. The solid support matrix can be magnetic beads, aflow-through matrix, or the material of a contraceptive device, such asa condom, diaphragm, cervical cap, vaginal ring, or sponge. In analternative embodiment, the solid support matrix can be an implantsuitable for surgical implantation in a host.

Conjugates can comprise more than one effector component that are thesame or different and can have the same or different effector functions.Diverse applications and uses of functional proteins and peptides, suchas the polypeptide of the invention attached to or immobilized on asolid support matrix and/or comprising polyethylene glycol conjugatedproteins or peptides in a review by Holmberg et al. (In Poly(EthyleneGlycol) Chemistry: Biotechnical and Biomedical Applications, Harris,ed., Plenum Press: New York (1992), pp. 303-324).

Antibodies

The invention also provides an antibody that recognizes (i.e., binds) apolypeptide of the invention. Preferably, the antibody binds to aportion or part of the amino acid sequence having the above-describedpercent sequence identity (e.g., 65% or greater, 70% or greater, 80% orgreater, 85% or greater, 90% or greater, 95% or greater, 96% or greater,97% or greater, 98% or greater, or 99% or greater) to SEQ ID NO: 1(e.g., binds to part of SEQ ID NO: 1 or SEQ ID NO: 2). In other words,it is preferable that the antibody binding site is part of the aminoacid sequence so defined. The availability of antibodies to any givenprotein is highly advantageous, as it provides the basis for a widevariety of qualitative and quantitative analytical methods, separationand purification methods, and other useful applications directed to thesubject proteins. Antibodies to the polypeptide of the invention can beprepared using well-established methodologies (e.g., such as themethodologies described in detail by Harlow and Lane, in Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,1988, pp. 1-725). Such antibodies can comprise both polyclonal andmonoclonal antibodies. Furthermore, such antibodies can be obtained andemployed either in solution-phase or by coupling the antibodies to adesired solid-phase matrix, such as magnetic beads or a flow throughmatrix. Such antibodies can be used in conjunction with well-establishedprocedures (e.g., such as described by Harlow and Lane (1988, supra))for the detection, quantification, or purification of a polypeptide ofthe invention or fusion protein or conjugate thereof or host celltransformed to produce the same. Such antibodies also can be used in thetreatment of a host as described further herein with respect to themethods of the invention.

Preferably, the antibody is anti-idiotypic in respect to gp120, i.e.,has an internal image of gp120 of a primate immunodeficiency virus.Preferably, the antibody can compete with gp120 of a primateimmunodeficiency virus for binding to a polypeptide of the invention(e.g., a polypeptide comprising SEQ ID NO: 1). In this regard, theprimary immunodeficiency virus preferably is HIV (e.g., HIV-1 or HIV-2).Anti-idiotypic antibodies can be generated in accordance with methodsknown in the art (see, for example, Benjamin, In Immunology: a shortcourse, Wiley-Liss, N.Y., 1996, pp. 436-437; Kuby, In Immunology, 3rded., Freeman, N.Y., 1997, pp. 455-456; Greenspan, et al., FASEB J., 7:437-443 (1993); and Poskitt, Vaccine, 9: 792-796 (1991)). For instance,a polypeptide of the invention (e.g., that can bind to gp120) can bedirectly administered to an animal, such that the animal generates andantibody that has an internal image of gp120. The production ofanti-idiotypic antibodies in an animal to be treated is known asanti-idiotype induction therapy, and is described, for example, byMadiyalakan et al. (Hybridoma, 14: 199-203 (1995)). Similarly, theantibody can be anti-idiotypic in respect to gp41.

Although nonhuman anti-idiotypic antibodies to gp120 are proving usefulas vaccine antigens in humans, in certain instances, the favorableproperties of an antibody of the invention might be further enhancedand/or any adverse properties further diminished, through humanizationstrategies, such as those reviewed by Vaughan (Nature Biotech., 16:535-539 (1998)).

Nucleic Acid

The invention provides a nucleic acid (e.g., an isolated or purifiednucleic acid) comprising a nucleotide sequence encoding a polypeptide ofthe invention as described herein. Thus, the invention provides anucleic acid comprising a nucleic acid sequence that encodes an aminoacid sequence that has about 65% or greater, 70% or greater, 75% orgreater, 80% or greater, 85% or greater, or 90% or greater sequenceidentity to SEQ ID NO: 1, or encodes the amino acid sequence SEQ ID NO:1 or SEQ ID NO: 2, or an antiviral fragment of SEQ ID NO: 1, providedthat the nucleic acid does not encode the amino acid sequence of SEQ IDNO: 4. The invention also encompasses a nucleic acid encoding apolypeptide or fusion protein comprising such an amino acid sequence,provided it does not encode the amino acid sequence of SEQ ID NO: 4. Theterm “nucleic acid” as used herein refers to a polymer of DNA or RNA(i.e., a polynucleotide), which can be single-stranded ordouble-stranded, synthesized or obtained from natural sources, and whichcan contain natural, non-natural, or altered nucleotides. Desirably, thenucleic acid of the invention encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, and preferably encodes the amino acidsequence SEQ ID NO: 1. A preferred nucleic acid comprises the nucleicacid sequence of SEQ ID NO: 3.

The invention also encompasses a nucleic acid comprising a nucleic acidsequence that is substantially identical to SEQ ID NO: 3. A sequence issubstantially identical to SEQ ID NO: 3 if it has about 65% or greater(e.g., about 65% or greater), preferably about 70% or greater (e.g.,about 75% or greater), such as about 80% or greater (e.g., about 85% orgreater) or even about 90% or greater (e.g., about 95% or greater)sequence identity to SEQ ID NO: 3.

Another indication that polynucleotide sequences are substantiallyidentical is if the first sequence and the complement of the secondsequence selectively hybridize to each other under stringent conditions.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 20° C. lower than the thermal melting point (Tm) for thespecific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Stringentconditions are defined, for the purposes of the invention, as thoseconditions that allow up to about 25% mismatch, more preferably up toabout 15% mismatch, and most preferably up to about 10% mismatch (e.g.,up to 5% mismatch). Hybridization and wash conditions that result insuch levels of stringency can be selected by the ordinarily skilledartisan using techniques known in the art.

One of ordinary skill in the art will appreciate, however, that twonucleic acid sequences can have substantially different sequences, yetencode substantially similar, if not identical, amino acid sequences,due to the degeneracy of the genetic code. The invention is intended toencompass such nucleic acids.

A variety of techniques used to synthesize the nucleic acids of thepresent invention are known in the art. See, for example, Lemaitre etal., PNAS USA, 84: 648-652 (1987).

The nucleic acid of the invention can be in the form of a vector. Inthis regard, the invention provides a vector comprising anabove-described nucleic acid. The vector can be targeted to acell-surface receptor if so desired. A nucleic acid as described abovecan be cloned into any suitable vector and can be used to transform ortransfect any suitable host. The selection of vectors and methods toconstruct them are commonly known to persons of ordinary skill in theart and are described in general technical references (see, in general,“Recombinant DNA Part D,” Methods in Enzymology, Vol. 153, Wu andGrossman, eds., Academic Press (1987)). Desirably, the vector comprisesregulatory sequences, such as transcription and translation initiationand termination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA or RNA. Preferably, the vector comprises regulatorysequences that are specific to the genus of the host. Most preferably,the vector comprises regulatory sequences that are specific to thespecies of the host.

Constructs of vectors, which are circular or linear, can be prepared tocontain an entire nucleic acid as described above or a portion thereofligated to a replication system functional in a prokaryotic oreukaryotic host cell. Replication systems can be derived from ColE1, 2mu plasmid, λ, SV40, bovine papilloma virus, and the like.

In addition to the replication system and the inserted nucleic acid, theconstruct can include one or more marker genes, which allow forselection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

One of ordinary skill in the art will appreciate that any of a number ofvectors known in the art are suitable for use in the invention. Suitablevectors include those designed for propagation and expansion or forexpression or both. Examples of suitable vectors include, for instance,plasmids, plasmid-liposome complexes, and viral vectors, e.g.,parvoviral-based vectors (i.e., adeno-associated virus (AAV)-basedvectors), retroviral vectors, herpes simplex virus (HSV)-based vectors,and adenovirus-based vectors. Any of these expression constructs can beprepared using standard recombinant DNA techniques described in, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989);Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y. (1994);Fischer et al., Transgenic Res., 9(4-5): 279-299 (2000); Fischer et al.,J. Biol. Regul. Homeost. Agents, 14: 83-92 (2000); deWilde et al., PlantMolec. Biol., 43: 347-359 (2000); Houdebine, Transgenic Research, 9:305-320 (2000); Brink et al., Theriogenology, 53: 139-148 (2000);Pollock et al., J. Immunol. Methods, 231: 147-157 (1999); Conrad et al.,Plant Molec. Biol., 38: 101-109 (1998); Staub et al., Nature Biotech.,18: 333-338 (2000); McCoimick et al., PNAS USA 96: 703-708 (1999);Zeitlin et al., Nature Biotech., 16: 1361-1364 (1998); Tacker et al.,Microbes and Infection, 1: 777-783 (1999); and Tacket et al., NatureMed., 4(5): 607-609 (1998). Examples of cloning vectors include the pUCseries, the pBluescript series (Stratagene, LaJolla, Calif.), the pETseries (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, Calif.).Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λ NM1149, also can be used. Examples of plant expressionvectors include pBI101, pBI101.2, pBI101.3, pBI121, and pBIN19(Clonetech, Palo Alto, Calif.). Examples of animal expression vectorsinclude pEUK-C1, pMAM, and pMAMneo (Clonetech).

An expression vector can comprise a native or normative promoteroperably linked to a nucleic acid as described above. The selection ofpromoters, e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the skill in the art. Similarly, thecombining of a nucleic acid as described above with a promoter also iswithin the skill in the art.

Isolated Cell

The nucleic acid of the invention can be introduced into a cell usingsuitable techniques (e.g., transfection, electroporation, transduction,micro-injection, transformation, and the like). Thus, the inventionprovides an isolated cell comprising the above-described nucleic acid ofthe invention. The nucleic acid can be in the form of a vector, which isoptionally targeted to a cell-surface receptor. Suitable host cellsinclude, but are not limited to an animal cells, such as a bird ormammalian cell, particularly a human cell, or a bacterial cell, such asE. coli (in particular E. coli TB-1, TG-2, DH5α, XL-Blue MRF′(Stratagene), SA2821, and Y1090), B. subtilis, P. aerugenosa, S.cerevisiae, and N. crassa. Preferably, the cell is a bacterium or yeast.A preferred bacterium is lactobacillus.

Compositions

The invention provides a composition (e.g., pharmaceutical composition)comprising (i) a polypeptide, fusion protein, conjugate, nucleic acid,or antibody of the invention (collectively referred to as the compoundsof the invention), as described herein, and (ii) a carrier, excipient,or adjuvant therefor. The amount of component (i) present in thecomposition will depend upon its end use. If the composition is to beused to inhibit a virus, then the composition should comprise anantiviral-effective amount of component (i). If the composition is to beused to induce an immune response in a host, then the composition shouldcomprise an amount of component (i) effective to induce the immuneresponse.

The carrier can be any of those conventionally used and is limited onlyby chemico-physical considerations, such as solubility and lack ofreactivity with the active agent (e.g., the compound of the invention),and by the route of administration. It is preferred that the carrier ispharmaceutically acceptable, and that the pharmaceutically acceptablecarrier is chemically inert to the active agent. Preferably, the carrierhas few or no detrimental side effects or toxicity under the conditionsof use. The pharmaceutically acceptable carriers described herein, forexample, vehicles, adjuvants, excipients, and diluents, are well-knownto those ordinarily skilled in the art and are readily available to thepublic. Typically, the composition, such as a pharmaceuticalcomposition, can comprise a physiological saline solution; dextrose orother saccharide solution; or ethylene, propylene, polyethylene, orother glycol.

The composition can further comprise at least one additional activeagent. The particular type of additional active agent will, of course,depend upon the end use of the composition. For instance, for theinhibition of a virus, the composition can comprise a substance (otherthan the compound of the invention) that is efficacious in inhibitingthe viral invection. If the composition is to be used to induce animmune response, the composition can further comprise an immunoadjuvant,such as polyphosphazene polyelectrolyte.

The composition can comprise more than one compound of the invention, orother pharmaceuticals, such as virucides, immunomodulators,immunostimulants, antibiotics, and absorption enhancers. Exemplaryimmunomodulators and immunostimulants include various interleukins,sCD4, cytokines, antibody preparations, blood transfusions, and celltransfusions. Exemplary antibiotics include antifungal agents,antibacterial agents, and anti-Pneumocystitis carnii agents. Exemplaryabsorption enhancers include bile salts and other surfactants, saponins,cyclodextrins, and phospholipids (Davis (1992), supra).

Method of Inhibiting Viral Infection

The compounds and compositions of the invention as described herein canbe used for any purpose. The polypeptide, fusion protein, and conjugateof the invention desirably can bind viral protein, such as gp41 andgp120, rendering these and other compounds and compositions of theinvention (e.g., the nucleic acid, antibody, and isolated cell) usefulfor investigating mechanisms of viral replication, infection, andinhibition. Also, the compounds and compositions of the invention areparticularly useful for antiviral purposes. The compounds andcompositions of the invention, for example, can be used to inhibit aviral infection in a host and can be used therapeutically orprophylactically. By therapeutically is meant that the host already hasbeen infected with the virus. By prophylactically is meant that the hosthas not yet been infected with the virus but is at risk of beinginfected with the virus. Prophylactic treatment is intended to encompassany degree of inhibition of viral infection, including, but not limitedto, complete inhibition, as one of ordinary skill in the art willreadily appreciate that any degree in inhibition of viral infection isadvantageous.

In this regard, the invention provides a method of inhibiting a viralinfection in a host, which method comprises administering to the host(e.g., a host comprising a viral infection) a viral infection-inhibitingamount of the polypeptide, fusion protein, conjugate, antibody, nucleicacid, isolated cell, or composition of the invention.

The invention also provides a method of inhibiting the infection of ahost by a Virus comprising administering to the host (e.g., a host thathas not been infected by the virus) an antibody of the invention in anamount sufficient to induce in the animal an immune response, whereuponthe infection of the animal with the virus is inhibited. Alternatively,or in addition to the administration of an antibody of the invention,the method can comprise the administration to the host of a polypeptide,fusion protein, or conjugate of the invention such that antibodies areproduced by the host. In yet another aspect, the method can comprise, inaddition to or instead of the administration of the antibody and/orpolypeptide, fusion protein, or conjugate, the administration of anucleic acid (or vector or isolated cell comprising the nucleic acid) tothe host, such that a polypeptide of the invention is produced by thehost, and, consequently antibodies to the polypeptide are produced inthe host.

The compounds and compositions of the invention (i.e., the polypeptide,fusion protein, conjugate, nucleic acid, isolated cell, antibody, andcompositions comprising same) useful in the inventive methods are aspreviously described herein.

The virus to be inhibited can be any suitable virus. Preferably, thevirus is a retrovirus, such as a primate immunodeficiency virus (e.g.,SIV) or HIV (e.g., HIV-1 or HIV-2). However, the invention can be usedto inhibit other viruses, as well (see, e.g., Principles of Virology:Molecular Biology, Pathogenesis, and Control, Flint et al., eds., ASMPress: Washington, D.C., 2000, particularly Chapter 19). Examples ofviruses include, but are not limited to one or more of the following:Type C and Type D retroviruses, HTLV-1, HTLV-2, FIV, FLV, MLV, BLV, BIV,equine infectious virus, anemia virus, bird viruses, such as birdinfluenza, SARS, and coronavirus, avian sarcoma viruses, such as Roussarcoma virus (RSV), hepatitis type A, B, non-A and non-B viruses,arboviruses, varicella viruses, human herpes virus (e.g., HHV-6),measles, mumps and rubella viruses, pox viruses, influenza viruses, suchas influenza viruses A and B, Ebola and other hemorrhagic fever viruses,and other viruses. Preferably, the virus to be inhibited comprises as asurface protein (e.g., a coat protein) a glycoprotein having a highmannose oligosaccharide, such as an immunodeficiency virus, in whichcase the host is preferably human, and the virus is HIV.

The above methods can further comprise the concurrent or pre- orpost-treatment with an adjuvant to enhance the antiviral effectivenessof the compounds and compositions of the invention, or to enhance theimmune response of the host. For example, the methods can furthercomprise the prior, simultaneous, or subsequent administration, by thesame or a different route, of a substance other than a compound orcomposition of the invention that is efficacious in inhibiting the virus(e.g., an antiviral agent) or inducing an immune response to the virus(e.g., an immunostimulant). See, for example, Harlow et al., 1988,supra.

With regard to the forgoing methods of the invention, the host can beany suitable animal, such as a bird (e.g., chicken, duck, goose, dove,and the like) or a mammal, such as a cow, horse, primate, pig, goat,cat, dog, rabbit, guinea pig, mouse, rat, hamster, guinea pig, or, mostpreferably, a human.

The dose administered to a host, such as an animal, in particular ahuman, in the context of the present invention should be sufficient toeffect a prophylactic or therapeutic response in the individual over areasonable time frame. The dose used to achieve a desired antiviralconcentration in vivo (e.g., 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 10 nM, 50 nM,100 nM, 200 nM, 400 nM, 500 nM, 600 nM, 800 nM, 1000 nM, or rangesthereof) will be determined by the potency of the particular activeagent employed, the severity of the disease state of the infectedindividual, as well as, in the case of systemic administration, the bodyweight and age of the infected individual. The size of the dose alsowill be determined by the existence of any adverse side effects that canaccompany the particular active agent employed. It is always desirable,whenever possible, to keep adverse side effects to a minimum. Thedosages of ddC and AZT used in AIDS or ARC patients are known in theart. A virustatic range of ddC is generally between 0.05 μM to 1.0 μM. Arange of about 0.005-0.25 mg/kg body weight is virustatic in mostpatients. The preliminary dose ranges for oral administration aresomewhat broader, for example, 0.001 to 0.25 mg/kg given in one or moredoses at intervals of 2, 4, 6, 8, 12, etc. hours. Currently, 0.01 mg/kgbody weight ddC given every 8 hrs is preferred. When given in combinedtherapy, the other antiviral agent, for example, can be given at thesame time as the present inventive active agent or the dosing can bestaggered as desired. The two drugs also can be combined in acomposition. Doses of each can be less when used in combination thanwhen either is used alone.

In terms of administration of the inventive antiviral agents orconjugates thereof, the dosage can be in unit dosage form, such as atablet or capsule. The term “unit dosage form” as used herein refers tophysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of acompound or composition of the invention, alone or in combination withother active agents, calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier, or vehicle.

The specifications for the unit dosage forms of the present inventiondepend on the particular compound of the invention employed and theeffect to be achieved, as well as the associated pharmacodynamics in thehost. The dose administered should be an antiviral effective amount oran amount necessary to achieve an effective level in the individualpatient.

Since the effective level is used as the preferred endpoint for dosing,the actual dose and schedule can vary, depending upon interindividualdifferences in pharmacokinetics, drug distribution, and metabolism. Theeffective level can be defined, for example, as the blood or tissuelevel (e.g., 0.1-1,000 nM) desired in the patient that corresponds to aconcentration of one or more active agents that inhibits a virus, suchas HIV, in an assay known to predict for clinical antiviral activity ofchemical compounds and biological agents. The effective level for agentsof the invention also can vary when the inventive active agent is usedin combination with other known active agents or combinations thereof.

One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired effectiveconcentration in the individual patient. One skilled in the art also canreadily determine and use an appropriate indicator of the effectiveconcentration of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

In the treatment of some virally infected individuals, it can bedesirable to utilize a mega-dosing regimen, wherein a large dose ofcompound or composition of the invention (i.e., active ingredient) isadministered, time is allowed for the active ingredient to take effect,and then a suitable reagent, device or procedure is administered to theindividual to inactivate or remove the active ingredient.

When the method comprises the administration of an isolated cell of theinvention to the host, the isolated cell is desirably a cell from anorganism that shares homology with the host or from the host itself,which has been previously isolated and contacted a nucleic acid of theinvention. Alternatively, the isolated cell is a nonpathogenic bacteriumor yeast. Preferably, the nonpathogenic bacterium is a lactobacillus.The contacting of a nucleic acid of the invention ex vivo with cellspreviously removed from a given animal, such as a mammal, in particulara human, in such a manner that the nucleic acid will become insertedinto the cell, is within the ordinary skill in the art. Such cellsexpress the polypeptide in vivo after reintroduction into the host. Thefeasibility of such a therapeutic strategy to deliver a therapeuticamount of the polypeptide in close proximity to the desired target cellsand pathogens, i.e., virus, more particularly retrovirus, specificallyHIV and its envelope glycoprotein gp120, has been demonstrated instudies with cells engineered ex vivo to express sCD4 (Morgan et al.(1994), supra). It is also possible that, as an alternative to ex vivoinsertion of a nucleic acid of the invention, such a nucleic acid can beinserted into cells directly in vivo, such as by use of an appropriateviral vector. Such cells transfected in vivo are expected to produceantiviral amounts of a polypeptide of the invention directly in vivo.

Alternatively, a nucleic acid of the invention can be inserted intosuitable nonmammalian cells, and such cells, when administered to ahost, will express therapeutic or prophylactic amounts of a polypeptide,fusion protein, or conjugate of the invention directly in vivo within oronto a desired body compartment of the host, in particular an animalsuch as a human. In a preferred embodiment of the present invention, amethod of female-controllable prophylaxis against viral infection, suchas HIV infection, comprises the intravaginal administration and/orestablishment of, in a female human, a persistent intravaginalpopulation of lactobacilli that have been transformed with a codingsequence of the present invention to produce, over a prolonged time,effective virucidal levels a polypeptide, a fusion protein, or conjugateof the invention directly on or within or onto the vaginal and/orcervical and/or uterine mucosa. It is noteworthy that both of the WorldHealth Organization (WHO), as well as the U.S. National Institute ofAllergy and Infectious Diseases, have pointed to the need fordevelopment of female-controlled topical microbicides, suitable forblocking the transmission of HIV, as an urgent global priority (Lange etal., Lancet, 341: 1356 (1993); and Fauci, NIAID News, Apr. 27, 1995).

The polypeptide, fusion protein, and conjugate of the invention comprisepeptides, and, as such, are particularly susceptible to hydrolysis ofamide bonds (e.g., catalyzed by peptidases) and disruption of essentialdisulfide bonds or formation of inactivating or unwanted disulfidelinkages (Carone et al., J. Lab. Clin. Med., 100:1-14 (1982)). There arevarious ways to alter molecular structure, if necessary, to provideenhanced stability to such compounds (Wunsch, Biopolymers, 22: 493-505(1983); and Samanen, in Polymeric Materials in Medication, Gebelein etal., eds., Plenum Press: New York (1985), pp. 227-242), which can beessential for preparation and use of pharmaceutical compositionscontaining such compounds for therapeutic or prophylactic applicationsagainst viruses, e.g., HIV. Possible options for useful chemicalmodifications include, but are not limited to, the following (adaptedfrom Samanen (1985), supra): (a) olefin substitution, (b) carbonylreduction, (c) D-amino acid substitution, (d) N-methyl substitution, (e)C-methyl substitution, (f) C—C′-methylene insertion, (g) dehydro aminoacid insertion, (h) retro-inverso modification, (I) N-terminal toC-terminal cyclization, and (j) thiomethylene modification.Polypeptides, fusion proteins, and conjugates also can be modified bycovalent attachment of carbohydrate and polyoxyethylene derivatives,which are expected to enhance stability and resistance to proteolysis(Abuchowski et al., in Enzymes as Drugs, Holcenberg et al., eds., JohnWiley New York (1981), pp. 367-378).

Other important general considerations for design of delivery strategysystems and compositions, and for routes of administration, for proteinand peptide drugs, such as those provided by the invention, also apply(see Eppstein, CRC Crit. Rev. Therapeutic Drug Carrier Systems, 5:99-139 (1988); Siddiqui et al., CRC Crit. Rev. Therapeutic Drug CarrierSystems, 3: 195-208 (1987); Banga et al., Int. J. Pharmaceutics, 48:15-50 (1988); Sanders, Eur. J. Drug Metab. Pharmacokinetics, 15: 95-102(1990); and Verhoef, Eur. J. Drug Metab. Pharmacokinetics, 15: 83-93(1990). The appropriate delivery system for a polypeptide, fusionprotein, or conjugate will depend upon its particular nature, theparticular clinical application, and the site of drug action. Especiallyin the case of oral delivery, but also possibly in conjunction withother routes of delivery, it can be desirable to use anabsorption-enhancing agent in combination with a given polypeptide,fusion protein, or conjugate. A wide variety of absorption-enhancingagents have been investigated and/or applied in combination with proteinand peptide drugs for oral delivery and for delivery by other routes(Verhoef, 1990, supra; van Hoogdalem, Pharmac. Ther., 44: 407-443(1989); Davis, J. Pharm. Pharmacol., 44(Suppl. 1): 186-190 (1992)). Mostcommonly, typical enhancers fall into the general categories of (a)chelators, such as EDTA, salicylates, and N-acyl derivatives ofcollagen, (b) surfactants, such as lauryl sulfate andpolyoxyethylene-9-lauryl ether, (c) bile salts, such as glycholate andtaurocholate, and derivatives, such as tauro-di-hydro-fusidate, (d)fatty acids, such as oleic acid and capric acid, and their derivatives,such as acylcarnitines, monoglycerides and diglycerides, (e)non-surfactants, such as unsaturated cyclic ureas, (f) saponins, (g)cyclodextrins, and (h) phospholipids.

Other approaches to enhancing oral delivery of protein and peptide drugsthat can be applied to the invention can include aforementioned chemicalmodifications to enhance stability to gastrointestinal enzymes and/orincreased lipophilicity. Alternatively, or in addition, the protein orpeptide drug can be administered in combination with other drugs orsubstances, which directly inhibit proteases and/or other potentialsources of enzymatic degradation of proteins and peptides. Yet anotheralternative approach to prevent or delay gastrointestinal absorption ofprotein or peptide drugs is to incorporate them into a delivery systemthat is designed to protect the protein or peptide from contact with theproteolytic enzymes in the intestinal lumen and to release the intactprotein or peptide only upon reaching an area favorable for itsabsorption. A more specific example of this strategy is the use ofbiodegradable microcapsules or microspheres, both to protect vulnerabledrugs from degradation, as well as to effect a prolonged release ofactive drug (Deasy, in Microencapsulation and Related Processes,Swarbrick, ed., Marcell Dekker, Inc.: New York (1984), pp. 1-60, 88-89,208-211). Microcapsules also can provide a useful way to effect aprolonged delivery of a protein and peptide drug after injection(Maulding, J. Controlled Release, 6: 167-176 (1987)).

There are numerous other potential routes of delivery of a protein orpeptide drug, such as the polypeptide, fusion protein, or conjugate ofthe invention. These routes include intravenous, intraarterial,intrathecal, intracisternal, buccal, rectal, nasal, pulmonary,transdermal, vaginal, ocular, and the like (Eppstein (1988), supra;Siddiqui et al. (1987), supra; Banga et al. (1988), supra; Sanders(1990), supra; Verhoef (1990), supra; Barry, in Delivery Systems forPeptide Drugs, Davis et al., eds., Plenum Press: New York (1986), pp.265-275; and Patton et al., Adv. Drug Delivery Rev., 8: 179-196 (1992)).With any of these routes, or, indeed, with any other route ofadministration or application, a protein or peptide drug. When such areaction is not desirable, it may be necessary to modify the molecule inorder to mask immunogenic groups. It also can be possible to protectagainst undesired immune responses by judicious choice of method offormulation and/or administration. For example, site-specific deliverycan be employed, as well as masking of recognition sites from the immunesystem by use or attachment of a so-called tolerogen, such aspolyethylene glycol, dextran, albumin, and the like (Abuchowski et al.(1981), supra; Abuchowski et al., J. Biol. Chem., 252: 3578-3581 (1977);Lisi et al., J. Appl. Biochem., 4: 19-33 (1982); and Wileman et al., J.Pharm. Pharmacol., 38: 264-271 (1986)). Such modifications also can haveadvantageous effects on stability and half-life both in vivo and exvivo. Procedures for covalent attachment of molecules, such aspolyethylene glycol, dextran, albumin and the like, to proteins aspreviously described herein are well-known to those skilled in the art,and are extensively documented in the literature (e.g., see Davis etal., In Peptide and Protein Drug Delivery, Lee, ed., Marcel Dekker: NewYork (1991), pp. 831-864).

Other strategies to avoid untoward immune reactions can also include theinduction of tolerance by administration initially of only low doses. Inany event, it will be apparent from the present disclosure to oneskilled in the art that the skilled artisan can select an advantageousstrategy from any of a wide variety of possible compositions, routes ofadministration, or sites of application.

The inventive compounds and compositions can be used in the context ofthe inventive method to inhibit viral infection as a result of sexualtransmission. When used for the prevention of sexual transmission ofviral infection (e.g., HIV infection), the method of the invention cancomprise vaginal, rectal, oral, penile, or other topical, insertional,or instillational treatment with (e.g., administration of) a viralinfection-inhibiting amount of a polypeptide, fusion protein, orconjugate of the invention, and/or other compound or composition of theinvention including viable host cells transformed to express such acompound, alone or in combination with one or more other antiviralagents. Antiviral agents used or being considered for use against sexualtransmission include, for example, nonoxynol-9 (Bird, AIDS, 5: 791-796(1991)), gossypol and derivatives (Polsky et al., Contraception, 39:579-587 (1989); Lin, Antimicrob. Agents Chemother., 33: 2149-2151(1989); and Royer, Pharmacol. Res., 24: 407-412 (1991)), and gramicidin(Bourinbair, Life Sci./Pharmacol. Lett., 54: PL5-9 (1994); andBourinbair et al., Contraception, 49: 131-137 (1994)).

Nonpathogenic commensal bacteria and yeasts also offer an attractivemeans of in situ delivery of a polypeptide, fusion protein, or conjugateof the invention to prevent sexual transmission of viral infections. Forexample, lactobacilli readily populate the vagina, and indeed are apredominant bacterial population in most healthy women (Redondo-Lopez etal., Rev. Infect. Dis., 12: 856-872 (1990); Reid et al., Clin.Microbiol. Rev., 3: 335-344 (1990); Bruce and Reid, Can. J. Microbiol.,34: 339-343 (1988); Reu et al., J. Infect. Dis., 171: 1237-1243 (1995);Hilier et al., Clin. Infect. Dis., 16(Suppl 4): S273-S281; and Agnew etal., Sex. Transm. Dis., 22: 269-273 (1995)). Lactobacilli are alsoprominent, nonpathogenic inhabitants of other body cavities, such as themouth, nasopharynx, upper and lower gastrointestinal tracts, and rectum.

It is well-established that lactobacilli can be readily transformedusing available genetic engineering techniques to incorporate a desiredforeign nucleic acid sequence, and that such lactobacilli can be made toexpress a corresponding desired foreign protein (see, e.g., Hols et al.,Appl. and Environ. Microbiol., 60: 1401-1413 (1994)). Therefore, withinthe context of the present disclosure, it will be appreciated by oneskilled in the art that viable host cells containing a nucleic acidsequence or vector of the invention, and/or expressing a polypeptide,fusion protein, or conjugate of the invention, can be used directly asthe delivery vehicle to the desired site(s) in vivo. Preferred hostcells for such delivery directly to desired site(s), such as, forexample, to a selected body cavity, can comprise bacteria. Morespecifically, such host cells can comprise suitably engineered strain(s)of lactobacilli, enterococci, or other common bacteria, such as E. coli,normal strains of which are known to commonly populate body cavities.More specifically, such host cells can comprise one or more selectednonpathogenic strains of lactobacilli, such as those described by Andreuet al. (1995, supra), especially those having high adherence propertiesto epithelial cells, such as, for example, adherence to vaginalepithelial cells, and suitably transformed using the nucleic acidsequences of the present invention.

As reviewed by McGroarty (FEMS Immunol. Med. Microbiol., 6: 251-264(1993)) the probiotic or direct therapeutic application of livebacteria, particularly bacteria that occur normally in nature, moreparticularly lactobacilli, for treatment or prophylaxis againstpathogenic bacterial or yeast infections of the urogenital tract, inparticular the female urogenital tract, is a well-established concept.However, inventive use of non-mammalian cells, particularly bacteria,more particularly lactobacilli, specifically engineered with a nucleicacid of the invention is heretofore unprecedented as a method oftreatment of an animal, specifically a human, to prevent infection by avirus, specifically a retrovirus, more specifically HIV-1 or HIV-2.

One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routecan be used to administer a particular drug, a particular route canprovide a more immediate and more effective reaction than another route.Furthermore, one skilled in the art will appreciate that the particularpharmaceutical carrier employed will depend, in part, upon theparticular active compound employed, and the chosen route ofadministration. Accordingly, there is a wide variety of suitableformulations of the compounds and compositions of the invention.

Formulations suitable for oral administration can consist of liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid, granules or freeze-dried cells; solutions orsuspensions in an aqueous liquid; oil-in-water emulsions or water-in-oilemulsions; lozenges comprising the active ingredient in a flavor,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert base, such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient ina suitable liquid carrier; as well as creams, emulsions, gels and thelike containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli or live lactobacillus culturesgenetically engineered to directly produce a polypeptide, fusionprotein, or conjugate of the invention, such carriers as are known inthe art. Tablet forms can include one or more of lactose, mannitol, cornstarch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscaimellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible carriers. Suitable formulations fororal delivery can also be incorporated into synthetic and naturalpolymeric microspheres, or other means to protect the agents of thepresent invention from degradation within the gastrointestinal tract(see, for example, Wallace et al., Science, 260: 912-915 (1993)).

The compounds and compositions of the invention, alone or in combinationwith other antiviral agents, can be made into aerosol formulations ormicroparticulate powder formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen and thelike.

The compounds and compositions of the invention, alone or in combinationwith other antiviral agents or modulators of absorption, can be madeinto suitable foimulations for transdermal application and absorption(Wallace et al. (1993), supra). Transdermal electroporation oriontophoresis also can be used to promote and/or control the systemicdelivery of the compounds and/or compositions of the present inventionthrough the skin (e.g., see Theiss et al., Meth. Find. Exp. Clin.Pharmacol., 13: 353-359 (1991)).

Formulations for rectal administration can be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate. Formulations suitable for vaginal administration can bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayformulas containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli or live lactobacillus culturesgenetically engineered to directly produce a polypeptide, fusionprotein, or conjugate of the invention, such carriers as are known inthe art to be appropriate. Similarly, the active ingredient can becombined with a lubricant as a coating on a condom. Indeed, preferably,the active ingredient is applied to and/or delivered by anycontraceptive device, including, but not limited to, a condom, adiaphragm, a cervical cap, a vaginal ring and a sponge.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The invention further provides a method of inhibiting a virus in abiological sample or in/on an inanimate object. The method comprisescontacting the biological sample or the inanimate object with aviral-inhibiting amount of a polypeptide, fusion protein, or conjugateof the invention, or compositions comprising the polypeptide, fusionprotein, or conjugate.

By viral-inhibiting amount is meant an amount of active agent, such asin the range of 0.1-1,000 nM (e.g., 0.5 nM, 1 nM, 10 nM, 50 nM, 100 nM,200 nM, 400 nM, 500 nM, 600 nM, 800 nM, or ranges thereof), sufficientto inhibit the virus so as to reduce, and desirably eliminate, itsinfectivity. The method optionally further comprises the prior,simultaneous or subsequent contacting, in the same manner or a differentmanner, of the biological sample or inanimate object with a substanceother than the polypeptide, fusion protein, or conjugate of theinvention that is efficacious in inhibiting the virus. The biologicalsample can be a cell, a tissue, an organ, a vaccine formulation, abodily fluid, and the like. Bodily fluids include blood and bloodproducts and components, semen (sperm), mucus membrane secretions,saliva, and the like. Preferably the virus that is inhibited isinfectious, such as HIV. The inanimate object can be a liquid or asolid. Solids include, for example, a surface, especially the surface ofan article of durable or consumable medical or laboratory equipment orsupply. Liquids include solutions, suspensions, emulsions, and the like,especially those used in a medical or laboratory setting. The methodalso can be used for the ex vivo virucidal sterilization of a biologicalsample or inanimate object for administration or implantation in apatient.

Formulations comprising a polypeptide, fusion protein, or conjugate ofthe invention suitable for virucidal (e.g., HIV) sterilization ofbiological samples or inanimate objects can be selected or adapted asappropriate, by one skilled in the art, from any of the aforementionedcompositions or formulations. Similarly, foimulations suitable for exvivo sterilization, or inhibition of virus in a biological sample or ina solution, suspension, emulsion, vaccine formulation or other material,which can be administered to a patient in a medical procedure, can beselected or adapted as appropriate by one skilled in the art, from anyof the aforementioned compositions or formulations. However, suitableformulations for sterilization or inhibition of virus in a biologicalsample or in/on an inanimate object are by no means limited to any ofthe aforementioned formulations or compositions. For example, suchformulations or compositions can comprise a polypeptide, fusion protein,or conjugate of the invention attached to a solid support matrix, tofacilitate contacting, or otherwise inhibiting infectious virus in abiological sample or inanimate object such as described above. The solidsupport matrix can comprise, for example, magnetic beads to facilitatecontacting and inhibition of infectious virus, and enable the subsequentmagnet-assisted removal of the beads from the biological sample orinanimate object. Alternatively, the solid support matrix can comprise acontraceptive device, such as a condom, a diaphragm, a cervical cap, avaginal ring or a sponge.

When a solid support matrix is used, the polypeptide or fusion proteinof the invention can be attached to the support matrix by any suitablemethod. For example, the polypeptide can be attached to the supportmatrix by use of an antibody to the polypeptide or fusion protein, or byconjugation of the polypeptide or fusion protein to at least oneeffector component suitable for binding to the support matrix. Effectorcomponents are previously discussed herein with respect to the conjugateof the invention. Methods of attaching an antibody to a solid supportmatrix are well-known in the art (see, for example, Harlow and Lane.Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory: ColdSpring Harbor, N.Y., 1988). Alternatively, the solid support matrix,such as magnetic beads, can be coated with streptavidin, in which thepolypeptide, fusion protein, or conjugate of the invention, isbiotinylated. The use of biotinylation as a means to attach a desiredbiologically active protein to a streptavidin-coated support matrix,such as magnetic beads, is well-known in the art.

Other methods of attaching a polypeptide, fusion protein, or conjugateto a solid support matrix can be used. For example, polyethylene glycolcan be used to attach such a compound to the matrix. When a polypeptide,fusion protein, or conjugate is attached to the matrix usingpolyethylene glycol, it is provided with a longer “tether” than would befeasible or possible for other attachment methods, such asbiotinylation/streptavidin coupling. A polypeptide, fusion protein, orconjugate of the invention coupled by a polyethylene glycol tether to asolid support matrix (such as magnetic beads, porous surface ormembrane, and the like) can permit optimal exposure of a bindingsurface, epitope, hydrophobic or hydrophilic focus, and/or the like, ofthe bound compound in a manner that, in a given situation and/or for aparticular virus, better facilitates inhibition of the virus.

Similarly, other types of solid support matrices can be used, such as amatrix comprising a porous surface or membrane, over or through which asample is flowed or percolated, thereby selectively inhibitinginfectious virus in the sample. The choice of solid support matrix,means of attachment to the solid support matrix, and means of separatingthe sample and the matrix-anchored compound will depend, in part, on thesample (e.g., fluid vs. tissue) and the virus to be inhibited. It isexpected that the use of a selected coupling molecule can confer certaindesired properties to a matrix that can have particularly advantageousproperties in a given situation.

The method of the invention also has utility in real time ex vivoinhibition of virus or virus infected cells in a bodily fluid, such asblood, e.g., in the treatment of viral infection, or in the inhibitionof virus in blood or a component of blood; for transfusion, in theinhibition or prevention of viral infection; in dialysis, such as kidneydialysis; and in inhibiting virus in sperm obtained from a donor for invitro and in vivo fertilization. The methods also have applicability inthe context of tissue and organ transplantations.

Matrix-anchored antibodies of the invention can be used in a method toinhibit virus in a sample. The antibody can be coupled to a solidsupport matrix using similar methods and with similar considerations asdescribed above for attaching polypeptides, fusion proteins, orconjugates to a solid support matrix. Preferably, the matrix is a solidsupport matrix, such as a magnetic bead or a flow-through matrix.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example illustrates viral envelope molecular target interactions ofSD1 (SEQ ID NO: 1).

The affinity of the SD1 polypeptide (SEQ ID NO: 1) and the scytovirinpolypeptide (SEQ ID NO: 4) for HIV envelope proteins gp120 and gp41 weredetermined using an ELISA protocol as described in O'Keefe et al., Mol.Pharmacol., 58: 982-992 (2000). For these demonstrations, ELISAprotocols were as follows. Briefly, the 100 ng/well of either gp120 andgp41 proteins was bound to a 96-well plate, which was then rinsed withPBS containing 0.05% Tween 20 (TPBS) and blocked with BSA. Between eachsubsequent step, the plate was again rinsed with TPBS (x3). The wellswere incubated with serial dilutions of scytovirin or SD1, followed byincubation with a 1:1000 dilution of anti-scytovirin rabbit polyclonalantibody preparation (see Boyd et al., Antimicrob. Agents Chemother.,41(7): 1521-1530 (1999)). The amount of bound scytovirin or SD1 wasdetermined by adding donkey-anti-rabbit antibodies conjugated tohorseradish peroxidase (1:2000 dilution, Amersham Life Sciences,Piscataway, N.J.). Upon addition of the horseradish peroxidase substratebuffer and color formation, the reaction was stopped by the addition of2M H₂SO₄ (after 5 minutes for the gp120 plate and 15 minutes for thegp41 plate) and absorbance was measured at 450 nm for each well.

Analysis of the data indicates that scytovirin and SD1 both appeared tobind gp120 and gp41 with approximately the same affinity (see FIGS. 1and 2). In contrast, when the same experiment was performed with SD2(residues 49-95 of scytovirin with a substitution of serine at position55), SD2 had significantly reduced binding (e.g., about 50% binding togp120) in comparison to scytovirin.

EXAMPLE 2

This example illustrates antiviral activity, in particular anti-HIVactivity, of a scytovirin as it compares to SD1.

An XTT-tetrazolium based assay was used to determine the anti-HIVactivity of scytovirin and SD1 against acute HIV-1 infection in CEM-SScells. The protocol followed was the same as described in Gulakowski etal., J. Virol. Methods, 33: 87-100 (1991).

SD1 and scytovirin showed comparable activity against the T-tropiclaboratory strain HIV-1_(RF) in CEM-SS cells with EC₅₀ values of 6.6 nMand 7.5 nM, respectively. Toxicity to the CEM-SS cell line was notdetected for either scytovirin or SD1 at concentrations up to 10,000 nM.In contrast, when the same experiment was performed with SD2 (residues49-95 of scytovirin with a substitution of serine at position 55), SD2displayed significantly lower anti-HIV activity than scytovirin with a40 fold higher EC₅₀ than scytovirin.

EXAMPLE 3

This example illustrates the anti-HIV activity and binding activity ofSD1(Cys7Ser) deletion mutants.

To examine whether the N- and C-terminus of SD1 are necessary forantiviral activity, a series of deletion mutants were constructed inwhich two, five, or ten amino acids were deleted from the N-terminus ofSD1, or eight amino acids were deleted from the C-terminus of SD1. Thedeletion mutants were subjected to the XTT-tetrazolium based assay asdescribed in Example 2.

Deletion of two, five, or ten N-terminal amino acids from SD1 completelyeliminated antiviral activity, indicating that N-terminal amino acids ofSD1 are necessary for maintaining the antiviral activity of SD1.Deletion of the eight amino C-terminal amino acids resulted in a 3- to7-fold decrease in anti-HIV potency, indicating that these C-terminalamino acids optimize the activity of SD1, but are not necessary in orderto retain some anti-viral activity.

To determine the gp120 and gp41 binding pattern of the SD1 deletionmutants, ELISA was performed as described in Example 1. The N-terminalmutants had almost no detectable binding activity with gp120 and gp41.The C-terminal mutant exhibited decreased binding activity as comparedto SD1.

EXAMPLE 4

This example illustrates that SD1 retains scytovirin's specificity tobind HIV-1 envelope glycoproteins through specific interactions withsubstructures of high mannose oligosachharides.

Scytovirnin specifically binds to a α-1-2, α1-2, α1-6-linkedtetramannoside, a substructure of oligomannose-8 and -9 found on HIVenvelope glycoproteins gp120 and gp41 (Adams et al., Chem. Biol., 11(6):875-881 (2004)). To determine whether or not SD1 retained this bindingspecificity, an ELISA experiment was performed to determine if thesugars could inhibit binding to HIV gp120. Briefly, a 96-well plate wasprepared as described in Example 1 with glycosylated gp120 or gp41 and100 ng/well of scytovirin or a scytovirin-derived peptide. Thescytovirin-derived peptides were (a) SD1, (b) SD1 with an 8 amino acidC-terminal truncation (SD1(1-40)), and (c) SD2. Each well also contained(a) various concentrations of oligomannose-8, (b) 100 μM of glucose,mannose, galactose, xylose, or N-acytylglucosamine, or (c) 100 μg/wellα-acid glycoprotein (to act as a carrier for sialic acid). The plate wasthen washed and visualized using anti-scytovirin polyclonal antibodiesas described in Example 1.

Glucose, mannose, galactose, xylose, N-acytylglucosamine, α-acidglycoprotein with sialic acid were not able to inhibit binding to gp120of SD1, SD1(1-40), or SD2, thereby indicating that the individualdomains of scytovirin retained some level of carbohydrate specificity.

This result was supported by additional experiments demonstrating thatoligomannose-8 was able to inhibit the binding of SD1 to gp41 in aconcentration-dependent manner. The hypothesis that scytovirinspecificity resided in the individual domains of scytovirin (representedby SD1 and SD2) also was confirmed by NMR analysis of a titration of thescytovirin-binding tetrasachharide (Adams et al., supra) intoscytovirin.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Any open-ended term (e.g.,comprising) used to describe the foregoing invention can be replacedwith a closed-ended term (e.g., consisting essentially of, or consistingof) without departing from the spirit and scope of the invention.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments will be apparent to those ofordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of inhibiting infection of a hostby a virus having a glycoprotein comprising a high mannoseoligosaccharide as a surface protein, which method comprisesadministering to the host a virus-inhibiting amount of a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, wherein the highmannose oligosaccharide is oligomannose-8, oligomannose-9, or acombination thereof.
 2. The method of claim 1, further comprising theprior, simultaneous, or subsequent administration, by the same route ora different route, of a substance other than the polypeptide that isefficacious in inhibiting the viral infection.
 3. The method of claim 1,wherein the virus is an immunodeficiency virus.
 4. The method of claim 3wherein the host is a human and the immunodeficiency virus is humanimmunodeficiency virus (HIV).
 5. The method of claim 1, wherein thepolypeptide further comprises a flexible spacer that links the aminoacid sequence of SEQ ID NO: 1 to a second amino acid sequence of SEQ IDNO:
 1. 6. The method of claim 1, wherein the polypeptide furthercomprises albumin.
 7. The method of claim 1, wherein the polypeptide ispart of a conjugate.
 8. The method of claim 7, wherein the conjugatefurther comprises one or more effector components selected from thegroup consisting of polyethylene glycol, dextran, an antiviral agent,and a solid support matrix.
 9. A method of inhibiting a virus having aglycoprotein comprising a high mannose oligosaccharide as a surfaceprotein in a biological sample or in/or an inanimate object, whichmethod comprises contacting the biological sample or the inanimateobject with a virus-inhibiting amount of a polypeptide comprising theamino acid sequence of SEQ ID NO:1, wherein the high mannoseoligosaccharide is oligomannose-8, oligomannose-9, or a combinationthereof.
 10. The method of claim 9, further comprising the prior,simultaneous, or subsequent contacting, in the same manner or in adifferent manner, of the biological sample or inanimate object with asubstance other than the polypeptide that is efficacious in inhibitingthe virus.
 11. The method of claim 9 wherein the biological sample is abodily fluid, a cell, a tissue, an organ, or a vaccine formulation. 12.The method of claim 9 wherein the polypeptide further comprises aflexible spacer that links the amino acid sequence of SEQ ID NO: 1 to asecond amino acid sequence of SEQ ID NO:
 1. 13. The method of claim 9wherein the polypeptide further comprises albumin.
 14. The method ofclaim 9 wherein the polypeptide is part of a conjugate.
 15. The methodof claim 14, wherein the conjugate further comprises one or moreeffector components selected from the group consisting of polyethyleneglycol, dextran, an antiviral agent, and a solid support matrix.